CN101265288A - Method for purifying CRM197 mutant - Google Patents

Method for purifying CRM197 mutant Download PDF

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CN101265288A
CN101265288A CN 200710013378 CN200710013378A CN101265288A CN 101265288 A CN101265288 A CN 101265288A CN 200710013378 CN200710013378 CN 200710013378 CN 200710013378 A CN200710013378 A CN 200710013378A CN 101265288 A CN101265288 A CN 101265288A
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ultrafiltration
crm197
protein
concentration
buffer
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CN 200710013378
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CN101265288B (en
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孙丽霞
薛 张
徐同文
王克波
王晶翼
婷 董
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齐鲁制药有限公司
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Abstract

The invention relates to a purification method of Escherichia coli expressed CRM197 mutant, which belongs to the technology field of medicine. The purification method includes the following steps: performing denaturation and renaturation of inclusion body, ultra-filtering for concentrating, performing chromatography using anion exchange for purifying, and colleting protein peak to obtain the high purity protein sample. The method is characterized in suitability for mass production, simple operation, short production period, high product purity, strong process stability, etc.

Description

CRM197突变体的纯化方法技术领域本发明涉及利用重组DNA技术生产基因工程药物,具体涉及大肠杆菌表达的CRM197突变体的纯化方法,属于医药技术领域。 TECHNICAL FIELD The purified CRM197 mutant of the present invention relates to the use of recombinant DNA technology to produce genetically engineered drugs, particularly relates to a method of purifying CRM197 mutant of E. coli expressed, belonging to the field of medical technology. 背景技术CRM197 (cross—reacting forms of toxin 197)是白喉毒素丧失了毒性的一种突变体,它是由野生型白喉毒素的碱基序列中由一个碱基G突变为A,从而导致了第52位氨基酸GLY突变为GLU。 Background CRM197 (cross-reacting forms of toxin 197) is the toxicity of diphtheria toxin lost a mutant, which is nucleotide sequence of the wild-type diphtheria toxin A by the mutation of a base G, resulting in 52 mutated amino acid GLY GLU. 从结构上看,CRM197具有完整的白喉毒素功能结构,但实验表明,NAD结合位点的变化影响了白喉毒素的酶活性及毒性。 From a structural point of view, CRM197 diphtheria toxin with a complete functional structure, but experiments show that changes in NAD binding sites affects the activity and toxicity of diphtheria toxin. 后来研究证明52位的GLY在白喉毒素NAD 结合位点起着重要作用,这就导致白喉毒素酶活性位点一一同NAD:EF2 ADP核糖转移酶结合区发生改变,导致CRM197片断A不能与EF2结合,不能对细胞起到毒性作用。 Later studies have shown that 52 of GLY diphtheria toxin plays in important role in NAD binding sites, which leads to the active site of diphtheria toxin with a NAD: EF2 ADP ribose-binding domain change, resulting in CRM197 A fragment can not be EF2 binding, can not play a toxic effect on cells. CRM197不具有酶活性以及毒性,但具有白喉毒素的免疫原性,因此CRM197也常被用作一种免疫蛋白载体交联其它半抗原一起作为疫苗。 CRM197 activity and having no toxicity, but have immunogenic diphtheria toxin, CRM197 and therefore also often used as a cross-linked immunogenic protein carrier as a vaccine together with other haptens. 早在1985年美国科学家就利用CRM197 的免疫原性,将嗜血流感菌表面的多糖交联到白喉类毒素及CRM197的蛋白载体上制作疫苗防治呼吸道感染,从防治效果上看,两种交联疫苗在效果上没有显著差异,但都能使小孩产生较强的免疫记忆。 As early as 1985, US scientists on the use of the immunogenicity of CRM197, the surface of the bacterium Haemophilus influenza polysaccharide cross-linked to the production of vaccine prevention of upper respiratory tract infections diphtheria toxoid and CRM197 carrier protein, the prevention effect from the point of view both pay combined vaccine no significant difference in the effect, but the child can produce a strong immune memory. 美国科学家针对小儿在2岁前对于肺炎球菌疫苗(球菌表面多糖, PnPs)不能产生免疫记忆,因此将球菌表面七价多糖交联于多种蛋白载体以便在小儿2岁前能对肺炎球菌产生抗体,经过多种蛋白载体交联后的动物及临床效果比较,发现PnPs 一CRM197能产生较好的免疫效果,而且安全无毒副作用。 US scientists can not produce children for immune memory for pneumococcal vaccine (cocci surface polysaccharides, PnPs) in the first 2 years old, so the seven-valent polysaccharide meningitidis surface cross-linking to a variety of carrier protein in order to produce antibodies against pneumococcus in children before the age of 2 , through a variety of animal protein carrier and Comparative clinical effects after crosslinking, a CRM197 PnPs was found to produce a better immune effect, safety and no toxic side effect. 目前该产品也已通过美国TOA批准上市,商品名为Prevnar。 The product has also been approved for marketing by the United States TOA, trade name Prevnar. 意大利科学家Francesco针对流行性脑膜炎也采用了利用其病菌表面多糖交联CRM197制作疫苗,他们从CRM197的结构上分析了交联的可行性,其中CRM197带有39个Lysin氨基酸残基和16个Arg残基,能提供较多的交联用的游离氨基。 Italian scientist Francesco meningococcal meningitis vaccine production also uses CRM197 bacteria with its surface polysaccharides crosslinked, they analyzed the feasibility of cross-linking structure from the CRM197, CRM197 wherein Lysin with 39 and 16 amino acid residues Arg residues, can provide more crosslinked with free amino groups. 从实验结果来看,虽然化学交联率较高,但都没有达到理论交联率。 From the experimental results, although higher chemical crosslinking rate, but have not reached the theoretical rate of crosslinking. 美国专利US6, 962, 803描述了白喉毒素CRM107采用棒状杆菌表达分泌到培养液中,得到的CRM107毒素收率虽然比较高,可高达培养基总蛋白的70%以上,但是由于培养基中的成分比较复杂,且盐离子强度较高,导致后续的纯化过程比较困难,工艺复杂,成本偏高, 目的产物收率低等。 U.S. Patent No. US6, 962, 803 describes the use of Corynebacterium diphtheria CRM107 toxin expressed and secreted into the medium, CRM107 toxin is a relatively high yield is obtained, the culture medium may be up to more than 70% of total protein, but since the medium ingredients complex, and the high ionic strength salt, resulting in more difficult the subsequent purification process, process complexity, high cost, low yield of the desired product. 针对现有CRM197的不足,海南天源康泽医药科技有限公司对原有C賜197的基因序列进行了改进,构建了表达CRM197的重组表达质粒并转化至大肠杆菌表达宿主中以包涵体形式表达。 The existing lack of CRM197, Hainan Kangze Pharmaceutical Co., Ltd. days source of the original gene sequence given 197 C has been improved, the recombinant expression plasmid constructed in CRM197 expression host and transformed into E. coli expressed inclusion bodies . 参见中国专利CN200610042194. 7。 See Chinese patent CN200610042194. 7. 发明内容对于采用大肠杆菌以包涵体形式表达重组CRM197突变体,本发明提供一种适宜于规模扩大化生产的快速、简便、高效的CRM197突变体的纯化方法,具有工艺简单、成本低, 目的产物收率高以及中间过程便于控制和易于规模化放大生产等特点。 SUMMARY OF THE INVENTION For use in E. coli expressing the recombinant CRM197 mutant form of inclusion bodies, the present invention provides a magnification suitable for the rapid production scale purification method is simple, efficient CRM197 mutant, simple process, low cost, the desired product intermediate and high yield process easy to control and easy to enlarged scale production and the like. 本发明CRM197突变体的纯化方法,包括如下步骤: (1)采用大肠杆菌以包涵体形式表达重组CRM197突变体,包涵体经过变、复性后得到含有目的蛋白组分的复性液;(2) 复性液通过超滤进行分离和浓縮,并用Tris-HCl缓冲液进行换液,将复性液浓縮5-25倍,得超滤浓縮液;(3) 上述超滤浓縮液通过阴离子交换层析进行纯化层析,截留核酸和大部分杂蛋白质,得到含有目的蛋白CRM197突变体的分级组分;(4) 用平衡缓冲液对阴离子交换层析系统进行平衡,使目的蛋白吸附到阴离子层析柱上,大部分杂质穿出阴离子层析柱;(5) 用添加了NaCl的平衡缓冲溶液对上述步骤(4)处理后的阴离子交换层析柱进行洗脱,使目的蛋白穿出阴离子交换层析柱,收集洗脱蛋白液,即得目的蛋白液。 The method of purifying CRM197 mutant of the present invention, comprising the steps of: (1) an E. coli expression as inclusion bodies using recombinant CRM197 mutant, variant inclusion body after renaturation to obtain refolding solution containing the protein component; (2 ) was refolded and concentrated by ultrafiltration, and the medium was changed with Tris-HCl buffer, renaturation was concentrated 5-25 fold by ultrafiltration to obtain a concentrate; (3) above ultrafiltration concentrate purification by chromatography, anion exchange chromatography, most of the entrapped nucleic acids and proteins heteroaryl, to obtain a classified component CRM197 mutant containing the protein; (4) anion-exchange chromatography equilibrated with equilibration buffer system, adsorbed target protein the anion exchange chromatography column, most of the impurities piercing anion chromatography column; (5) supplemented with NaCl in the equilibration buffer solution of the above step (4) after processing by anion exchange chromatography column eluted target protein through an anion exchange chromatography column, collecting eluted protein solution, i.e., to obtain the protein solution. 经过上述步骤的处理,得到的目标蛋白CRM197突变体的纯度高, 一次处理量大,且收率大大提高,为大规模生产提供了技术基础。 After processing the above steps, a high purity of CRM197 mutant protein obtained, a processing capacity, and greatly improving the yield, providing the basis for mass production technology. 上述步骤(1)中的变、复性方法可采用蛋白变、复性通用的方法。 Varying the above step (1), renaturation protein variant employed, renaturation general method. 上述步骤(2)中所述的复性液通过超滤进行分离和浓縮,可一次性进行超滤浓縮, 也可先超滤去除大分子蛋白或杂质,再进行超滤浓縮。 The above step (2) in the refolding solution is concentrated by ultrafiltration and may be disposable ultrafiltration, ultrafiltration to remove macromolecules may be proteins or impurities, and then concentrated by ultrafiltration. 在层析前加上超滤浓縮步骤,除去复性液中的大、小分子蛋白、多肽或其它杂质,以利于后续步骤的操作。 Before chromatography coupled with ultrafiltration step, to remove large liquid refolding, small proteins, polypeptides or other contaminants, to facilitate the operation of subsequent steps. 上述步骤(3)中所述的阴离子交换层析,所使用的阴离子交换层析柱的层析介质选自琼脂糖凝胶(S印harose)类或聚苯乙烯(SOURCE)类,配基选自季铵基(Q)或二乙基氨乙基(DEAE)。 Chromatography medium is selected from agarose gels (S printed harose) of the polystyrene-based or (3) in the anion exchange chromatography step, anion exchange chromatography column is used (the SOURCE) class ligand selected from the group since quaternary ammonium groups (Q) or diethylaminoethyl (DEAE). 所使用的阴离子交换层析介质具体选自下列之一:DEAE-S印harose FF, QS印harose FF, S0URCE30-Q或Q-Big-Beds,最优选DEAE-S印harose FF层析介质。 Anion exchange chromatography using specific media is selected from one of the following: DEAE-S plate harose FF, QS printing harose FF, S0URCE30-Q or Q-Big-Beds, most preferably DEAE-S plate harose FF chromatographic medium. 上述步骤(4)和(5)中的平衡缓冲液选用Tris-HCl缓冲液或磷酸盐缓冲液,缓冲液的浓度为10腿ol/L-lmol/L;缓冲液的pH值为6. 0-11. 0。 The above step (4) and (5) the selection of the equilibration buffer Tris-HCl buffer or phosphate buffer, the buffer is a concentration of the leg 10 ol / L-lmol / L; pH 6.0 buffer value -11 0. 上述步骤(5)中所述的添加了NaCl的平衡缓冲液,其中NaCl浓度为10mmol/L—lmol/L。 The above step (5) in the equilibration buffer was added the NaCl, wherein the NaCl concentration of 10mmol / L-lmol / L. 上述步骤(5)中的洗脱方式可以采用非梯度洗脱或连续梯度洗脱。 The above step (5) in a non-eluting ways or continuous gradient elution gradient employed. 本发明采用大肠杆菌以包涵体形式表达重组CRM197突变体,如CN200610042194. 7"白喉毒素突变体CRM197及其制备方法"中提供的包涵体,进行进一步纯化。 The present invention employs E.coli inclusion body recombinant CRM197 mutant as CN200610042194. 7 "CRM197 diphtheria toxin mutant and its preparation method 'provided in the form of inclusion body further purification. 本发明的CRM197突变体的纯化方法的技术关键点在于:在对复性液进行离子交换层析前,先用超滤进行了分离和浓縮,使目的蛋白的含量提高,使后面的纯化过程更简单,节约了成本,提高了收率。 CRM197 mutant purification method of the present invention in the art key body comprising: prior to refolding by ion exchange chromatography was first performed using the separation and concentration by ultrafiltration, the content of protein is increased, so that the back of the purification process simpler, cost savings, increasing the yield. 通过本发明的方法制备的CRM197突变体收率高、纯度好,可用于疫苗方面的交联使用; 同时采用本发明的方法纯化CRM197突变体,工艺简便、快速,设备投资小,规模放大容易, 适合大规模生产。 CRM197 mutant yield produced by the process of the present invention, the purity, and can be used for crosslinking of vaccines; simultaneous purification of CRM197 mutant, the method of the present invention the process is simple, fast, small investment, easy to scale up, suitable for mass production. 附图说明图1是利用SDS-PAGE电泳分析本发明方法纯化后的CRM197突变体纯度图谱。 BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a CRM197 mutant analysis method of the present invention after purification by SDS-PAGE purity of the electrophoresis pattern. 图中的l、 2、 3分别对应为三次实验的阴离子DEAE层析收集液的SDS-PAGE凝胶电泳图谱。 FIG. L, 2, 3 correspond to SDS-PAGE gel electrophoresis pattern of an anionic DEAE chromatography were collected for three experiments. 具体实施方式以下通过实施例将进一步说明本发明,这些实施例不应作为本发明的限制。 DETAILED DESCRIPTION The present invention will be further described hereinafter, the present invention should not be limited to these embodiments by Examples. 本发明的方法包括顺序使用包涵体的变、复性,复性液的超滤分离,阴离子交换层析来纯化CRM197突变体。 The method of the present invention comprises the use of sequence variants of inclusion bodies, refolding, refolding ultrafiltration liquid separation, purified CRM197 mutant anion exchange chromatography. CRM197突变体包涵体的制备参见CN200610042194. 7"白喉毒素突变体CRM197及其制备方法"实施例1中2. 4"采用超声破碎的方法提取包涵体蛋白,经诱导表达后的菌液以5000Xg离心10min,然后用缓冲液(50誦ol/LTris-HC1、 5mmol/LEDTA)重悬,用超声破碎仪按1.25A脉冲破碎10min,然后离心收集沉淀,用缓冲液(50mmol/L Tris — HC1、 5mmol/L EDTA)洗包涵体洗8遍,离心收集沉淀得到包涵体。"以下实施例中使用的超滤器是Millipore公司的Pellicon-2,超滤膜采用盒式膜堆。 CRM197 mutant preparation see inclusions CN200610042194. 7 "diphtheria toxin mutant CRM197 and preparation method" in Example 1 2.4 "extraction method using sonication inclusion body protein, expression induced by bacteria after centrifugation at 5000Xg 10min, then with buffer (50 recite ol / LTris-HC1, 5mmol / LEDTA) was resuspended by a sonicator broken 1.25A pulse 10min, then collected by centrifugation with buffer (50mmol / L Tris - HC1, 5mmol ultrafilter used in the examples / L EDTA) washing inclusion bodies washed 8 times, inclusion bodies collected by centrifugation. "the following are embodiments of the company Millipore Pellicon-2, using ultrafiltration membrane cartridge stack. 实施例l1、 CRM197突变体包涵体的溶解和变、复性10g CRM197突变体包涵体加入1000ml变性液中,所用变性液含8mol/L尿素,lml(3-巯基乙醇,Tris-HC1 50隱ol/L, pH8,0,搅拌溶解30min,离心力为15000Xg—29000Xg离心10min,收集上清,倒入10L Tris-HCl缓冲液中,搅拌混匀后于4-10。C复性24-72小时。2、 复性液的超滤分离和浓縮先用pH为7.0,浓度为10mmol/L的Tris-HC1缓冲溶液对超滤器和大孔径(300KD)超滤膜进行平衡,保持进出口压力分别为1.3bar和0.3bar,开始进行大分子蛋白或杂质的截留浓縮。当复性液浓縮至1L左右时;用1 —3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次,收集超滤透过液。将大孔径(300kd)超滤膜堆换为小孔径(10kd) 超滤膜堆,按照上述方法将上述超滤透过液进行浓縮,浓縮至1L左右时,用2倍体积的上述Tris-HC1缓冲液稀释后继续超滤至相同的 Example l1, CRM197 mutant variants and inclusion bodies were dissolved, renatured inclusion bodies were added 10g CRM197 mutant 1000ml denaturing solution, the denaturing solution containing 8mol / L urea, lml (3- mercaptoethanol, Tris-HC1 50 used implicitly ol / L, pH8,0, dissolved with stirring 30min, centrifugal force 15000Xg-29000Xg centrifugal 10min, the supernatant was collected, poured into 10L Tris-HCl buffer solution, mixed with stirring for 24-72 hours after 4-10.C renaturation. 2, separation and concentration by ultrafiltration refolding solution to a pH of 7.0, a concentration of 10mmol / L of Tris-HC1 buffer solution of ultrafiltration and a large aperture (300 kD) ultrafiltration membrane balance and maintain outlet pressure, respectively of 1.3bar and 0.3bar, macromolecular protein concentrate interception started or when impurities refolding solution was concentrated to about 1L;. with the above buffer solution refolding volumes of 1-3, and then to the same ultrafiltration volume; thus diluted 2 times, collecting a large pore ultrafiltration permeate (300 kD) membrane stack changed to small aperture (10 kD) ultrafiltration membrane reactor, according to the above method described above was concentrated by ultrafiltration permeate. when concentrated to approximately 1L, the above-described diluted with Tris-HC1 buffer, 2 volumes of the same continue to ultrafiltration 积,共稀释3次,收集浓縮液。通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到85%以上。3、 阴离子交换层析用300ml浓度为10mmol/L, pH为7. 0的Tris-HC1平衡缓冲液平衡DEAE-S印harose FF (L6X15cm)层析柱,将超滤后的浓縮液上柱。4、 平衡、洗脱、、过滤用浓度为10mmol/L, pH为7.0的Tris-HC1平衡缓冲液冲洗3个柱体积,换用含有0.5mol/LNaCl的pH为7. 0、浓度为10mmol/L Tris-HCl缓冲液作为洗脱液进行洗脱,收集洗脱蛋白液。所得的目的蛋白液中CRM197突变体的级分浓度可达到8mg/ml以上,纯度达到98%以上。 实施例21、 CRM197突变体包涵体的溶解和变、复性,同实施例l。2、 复性液的超滤分离和浓縮先用pH为8.0,浓度为30腿ol/L的Tris-HCl缓冲溶液对超滤器和大孔径(300KD)超滤膜进行平衡,保持进出口压力分别为1.3bar和0.3bar,开始进行大分子蛋白或杂质的截留浓縮。当复 Product were diluted 3 times, collecting the concentrate. Content concentration level CRM197 mutant collected and concentrated by ultrafiltration up to 3mg / ml, more than 85% purity of .3, anion-exchange chromatography with 300ml concentration of 10mmol / L, pH of the equilibration buffer Tris-HC1 equilibrium DEAE-S is printed harose FF 7. 0 (L6X15cm) column, the column .4, balance the concentrate after ultrafiltration, the concentration of eluted ,, filtered of 10mmol / L, pH of the equilibration buffer Tris-HC1 7.0 rinsed 3 column volumes, for a pH containing 0.5mol / LNaCl to 7.0, at a concentration of 10mmol / L Tris-HCl buffer as eluent for elution, the protein was eluted Sectional concentration level of the protein solution obtained in CRM197 mutant can reach more than 8mg / ml, more than 98% purity. Example 21 is, CRM197 mutant and variant embodiments inclusion bodies were dissolved, complex resistance, in Example L.2, ultrafiltration separation and concentration of the refolding solution to a pH of 8.0, a concentration of the leg 30 ol / L of Tris-HCl buffer solution to ultrafiltration and a large aperture (300 kD) ultrafiltration balance cutoff membrane, to maintain outlet pressure 1.3bar and 0.3bar respectively, begin macromolecular protein or impurities concentrated. when the complex 缓冲液浓縮至1L左右时;用1—3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次,收集超滤透过液。将大孔径(300kd)超滤膜堆换作小孔径(10kd) 超滤膜堆,按照上述方法将上述超滤透过液进行浓缩,浓縮至1L左右时,用2倍体积的上述Tris-HCl缓冲液稀释后继续超滤至相同的体积,共稀释3次,收集浓縮液。 The buffer solution was concentrated to about 1L; with the above buffer solution Refolding volumes of 1-3, and then to the same ultrafiltration volume; thus diluted 2 times, collecting a large pore ultrafiltration permeate (300kd). after the membrane stack change for small aperture (10 kD) ultrafiltration membrane reactor, according to the method described above was concentrated by ultrafiltration permeate above, when concentrated to about 1L, diluted with 2 volumes of the Tris-HCl buffer continues ultrafiltration to the same volume, were diluted 3 times, collecting the concentrate. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到80%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / ml, more than 80% purity. 3、 阴离子交换层析用300ml浓度为100mmol/L, pH为8. 0的Tris-HC1平衡缓冲液平衡QS印harose FF (1. 6 X15cm)层析柱,将超滤后的浓縮液上柱。 3, anion-exchange chromatography with 300ml concentration of 100mmol / L, pH to 8.0 of Tris-HC1 equilibrated with equilibration buffer QS printing harose FF (1. 6 X15cm) column, on the concentrate after ultrafiltration column. 4、 平衡、洗脱、过滤用浓度为100ram?l/L, pH为8.0的Tris-HCl平衡缓冲液冲洗3个柱体积,换用含有0.2mol/L NaCl的pH'为8.0、浓度为100mmol/L Tris-HCl缓冲液作为洗脱液进行洗脱,收集洗脱蛋白液。 4, balance, elution with a concentration filtered 100ram? L / L, pH rinsed with 3 column volumes of equilibration buffer Tris-HCl 8.0, a pH change containing 0.2mol / L NaCl 'of 8.0, a concentration of 100mmol / L Tris-HCl buffer and eluted as eluant eluted protein was collected. 所得的目的蛋白液中CRM197突变体的级分浓度可达到8mg/ml以上,纯度达至1」98%以上。 Level of the protein solution resulting mutant CRM197 content concentration can reach more than 8mg / ml, or more purity to "98%. 实施例31、 CRM197突变体包涵体的溶解和变、复性,同实施例l。 Embodiment 31, CRM197 mutant variants and inclusion bodies were dissolved, renatured embodiment, with the embodiment of Example l. 2、 复性液的超滤分离和浓縮先用pH为9.0,浓度为50腿ol/L的Tris-HC1缓冲溶液对超滤器和和大孔径(500KD)超滤膜进行平衡,保持进出口压力分别为1.3bar和0.3bar,开始进行大分子蛋白或杂质的截留浓縮。 2, separation and concentration by ultrafiltration refolding solution to a pH of 9.0, a concentration of the leg 50 ol / L of Tris-HC1 buffer solution and of the ultrafilter and a large aperture (500 kD) ultrafiltration membrane balance, into the holding outlet pressures of 1.3bar and 0.3bar, macromolecular protein concentrate interception started or impurities. 当复性缓冲液浓縮至1L左右时;用1—3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次,收集超滤透过液。 When the refolding buffer is concentrated to about 1L; with the above buffer solution Refolding volumes of 1-3, and then to the same ultrafiltration volume; thus diluted 2 times, ultrafiltration permeate collected. 将大孔径(500kd)超滤膜堆换作小孔径(5kd)超滤膜堆,按照上述方法将上述超滤透过液进行浓縮,浓縮至1L左右时,用2倍体积的上述Tris-HCl缓冲液稀释后继续超滤至相同的体积,共稀释3次,收集浓縮液。 When the large pore size (500 kD) membrane stack change for small aperture (5kD) ultrafiltration membrane reactor, according to the method described above was concentrated through ultrafiltration above was concentrated to approximately 1L, the above volumes of Tris 2 -HCl buffer, diluted to the same volume of ultrafiltration continues, a total of 3 times diluted, the concentrated solution was collected. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到80%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / ml, more than 80% purity. 3、 阴离子交换层析用300ml pH为9.0,浓度为50腿o1/ L的Tris-HC1平衡缓冲液平衡S0URCE30-Q (1.6 X15cm)层析柱,将超滤后的浓縮液上柱。 3, anion exchange chromatography using 300ml pH 9.0, at a concentration of 50 leg o1 / L of Tris-HC1 equilibrated with equilibration buffer (1.6 X15cm) S0URCE30-Q chromatography column, the column was the concentrate after ultrafiltration. 4、 平衡、洗脱、过滤用浓度为50mrao1/ L, pH为9. 0的Tris-HC1平衡缓冲液冲洗3个柱体积,然后将NaCl 的浓度在20min内从0腿ol/L升到100隱ol/L,继续在100min内将NaCl浓度从100mmol/L升高到lmol/L进行梯度洗脱,收集洗脱蛋白液。 4, balance, elution with a concentration filtered 50mrao1 / L, pH washed three column volumes of Tris-HC1 9.0 equilibration buffer, and then the NaCl concentration in 20min rise from 0 leg 100 ol / L implicit ol / L, will continue within 100min NaCl concentration increased from 100mmol / L to lmol / L gradient elution, the protein was eluted. 所得的目的蛋白中CRM197突变体的纯度大于98%,浓度达到10mg/ml以上。 The purity of CRM197 mutant protein in greater than 98%, a concentration of more than 10mg / ml. 实施例41、 CRM197突变体包涵体的溶解和变、复性,同实施例l。 Embodiment 41, CRM197 mutant variants and inclusion bodies were dissolved, renatured embodiment, with the embodiment of Example l. 2、 复性液的超滤分离和浓縮先用pH为6.0,浓度为10mmol/L的磷酸盐缓冲溶液对超滤器和小孔径(10kd)超滤膜进行平衡,保持进出口压力分别为1.3bar和0.3bar,进行复性液的浓縮。 2, separation and concentration by ultrafiltration refolding solution to a pH of 6.0, a concentration of 10mmol / L phosphate buffer solution and of the small pore size ultrafilter (10 kD) ultrafiltration membrane balance, outlet pressure were held 1.3bar and 0.3bar, concentrated renaturation solution. 当复性液浓縮至1L左右时;用1一3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次, 收集超滤透过液。 When refolding solution was concentrated to about 1L; refolding of the buffer solution was washed with 3 volumes of a 1, then the same volume of ultrafiltration; thus diluted 2 times, ultrafiltration permeate collected. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到80%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / ml, more than 80% purity. 3、 阴离子交换层析, 用300ml浓度为10mmol/L, p服.0的磷酸盐平衡缓冲液平衡DEAE-S印harose FF (1.6X15cm)层析柱,将超滤后的浓縮液上柱。 3, anion exchange chromatography, concentration with 300ml of 10mmol / L, p .0 serving phosphate equilibration buffer equilibrium DEAE-S plate harose FF (1.6X15cm) column, the column was the concentrate after ultrafiltration . 4、 平衡、洗脱、过滤用浓度为10mraol/L, pH6. 0的磷酸盐平衡缓冲液冲洗3个柱体积,然后将NaCl的浓度在20min内从0 mmol/L升到10Ommol/L,继续在100min内将NaCl浓度从100mmol/L升高到lmol/L进行连续梯度洗脱,收集洗脱蛋白液。 4, balance, elution with a concentration filtered 10mraol / L, pH6. 0 phosphate equilibration buffer flushed with 3 column volumes, then the NaCl concentration was raised in 20min 10Ommol / L from 0 mmol / L, continue 100min in the NaCl concentration increased from 100mmol / L to lmol / L continuous gradient elution, eluted protein was collected. 所得的目的蛋白中CRM197突变体的纯度大于98%,浓度达到10mg/ml以上。 The purity of CRM197 mutant protein in greater than 98%, a concentration of more than 10mg / ml. 实施例51、 CRM197突变体包涵体的溶解和变、复性,同实施例l。 Example 51, was dissolved and the CRM197 mutant variations, renaturation of inclusion bodies, with the embodiment of Example l. 2、 复性液的超滤分离和浓縮先用pH为7.0,浓度为50mmol/L磷酸盐缓冲溶液对超滤器和大孔径(500kd)超滤膜进行平衡,保持进出口压力分别为l. 3bar和0. 3bar,开始进行大分子蛋白或杂质的截留浓縮。 2, separation and concentration by ultrafiltration refolding solution to a pH of 7.0, a concentration of 50mmol / L phosphate buffer solution to ultrafiltration and a large aperture (500 kD) ultrafiltration membrane balance, l were maintained outlet pressure . 3bar and 0. 3bar, macromolecular protein concentrate starts entrapping or impurities. 当复性缓冲液浓縮至1L左右时;用1一3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次,收集超滤透过液。 When the refolding buffer is concentrated to approximately 1L; refolding of the buffer solution was washed with 3 volumes of a 1, then the same volume of ultrafiltration; thus diluted 2 times, ultrafiltration permeate collected. 将大孔径(500kd)超滤膜堆换作小孔径(10kd) 超滤膜堆,按照上述方法将上述超滤透过液进行浓縮,浓縮至1L左右时,用2倍体积的上述磷酸盐缓冲液稀释后继续超滤至相同的体积,共稀释3次,收集浓縮液。 When the large pore size (500 kD) membrane stack change for small aperture (10 kD) ultrafiltration membrane reactor, according to the method described above was concentrated through ultrafiltration above was concentrated to approximately 1L, the phosphoric acid with 2 volumes of after dilution ultrafiltration continues salt buffer to the same volume, it was diluted 3 times, collecting the concentrate. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到85%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / ml, more than 85% purity. 3、 阴离子交换层析用300ml浓度为50mraol/L, pH为7. 0的磷酸盐平衡缓冲液平衡Q-Big-Beds(l. 6X 15cm) 层析柱,将超滤后的浓縮液上柱。 3, anion-exchange chromatography with 300ml concentration 50mraol / L, pH 7.0 phosphate buffer balanced equilibrium Q-Big-Beds (l. 6X 15cm) column, on the concentrate after ultrafiltration column. 4、 平衡、洗脱、过滤用浓度为50咖ol/L, pH为7.0的磷酸盐平衡缓冲液冲洗3个柱体积,换用含有20mmol/L NaCl的pH为7.0、浓度为50mmol/L磷酸盐缓冲液作为洗脱液进行洗脱,收集洗脱蛋白液。 4, balance, eluting coffee filter at a concentration of 50 ol / L, pH rinsed with 3 column volumes of equilibration buffer phosphate 7.0, a pH change containing 20mmol / L NaCl 7.0, at a concentration of 50mmol / L phosphate salt buffer as eluent elution, the protein was eluted. 所得的目的蛋白液中CRM197突变体的级分浓度可达到8mg/ml以上,纯度达到98%以上。 Level of the protein solution resulting mutant CRM197 content concentration can reach more than 8mg / ml, more than 98% purity. 实施例61、 CRM197突变体包涵体的溶解和变、复性,同实施例l。 Example 61, was dissolved and the CRM197 mutant variations, renaturation of inclusion bodies, with the embodiment of Example l. 2、 复性液的超滤分离和浓縮先用pH为8.0,浓度为100mmol/L的磷酸盐缓冲溶液对超滤器和和大孔径(500KD)超滤膜进行平衡,保持进出口压力分别为1.3bar和0.3bar,开始进行大分子蛋白或杂质的截留浓縮。 2, separation and concentration by ultrafiltration refolding solution to a pH of 8.0, a concentration of 100mmol / L phosphate buffer solution and of the ultrafilter and a large aperture (500 kD) ultrafiltration membrane balance and maintain outlet pressure, respectively of 1.3bar and 0.3bar, macromolecular protein concentrate interception started or impurities. 当复性缓冲液浓縮至1L左右时;用1一3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次,收集超滤透过液。 When the refolding buffer is concentrated to approximately 1L; refolding of the buffer solution was washed with 3 volumes of a 1, then the same volume of ultrafiltration; thus diluted 2 times, ultrafiltration permeate collected. 将大孔径(500kd)超滤膜堆换作小孔径(5kd)超滤膜堆,按照上述方法将上述超滤透过液进行浓縮,浓縮至1L左右时,用2倍体积的上述磷酸盐缓冲液稀释后继续超滤至相同的体积,共稀释3次,收集浓縮液。 When the large pore size (500 kD) membrane stack change for small aperture (5kD) ultrafiltration membrane reactor, according to the method described above was concentrated through ultrafiltration above was concentrated to approximately 1L, the phosphoric acid with 2 volumes of after dilution ultrafiltration continues salt buffer to the same volume, it was diluted 3 times, collecting the concentrate. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到80%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / ml, more than 80% purity. 3、 阴离子交换层析用300ml浓度为100誦ol/L, pH为8. 0的磷酸盐平衡缓冲液平衡QS印harose FF (1.6 X15cm)层析柱,将超滤后的浓縮液上柱。 3, anion-exchange chromatography with 300ml recite a concentration of 100 ol / L, pH 8.0 phosphate buffer balanced equilibrium QS printing harose FF (1.6 X15cm) column, the column was the concentrate after ultrafiltration . 4、 平衡、洗脱、过滤用浓度为100mmol/L, pH为8. 0的磷酸盐平衡缓冲液冲洗3个柱体积,换用含有O. 3mol/L NaCl的pH为8.0、浓度为100mmol/L磷酸盐缓冲液作为洗脱液进行洗脱,收集洗脱蛋白液。 4, balance, elution with a concentration filtered 100mmol / L, pH of the equilibration buffer 8.0 phosphate wash with 3 column volumes, containing a pH change O. 3mol / L NaCl 8.0, at a concentration of 100mmol / L phosphate buffer and eluted as eluant eluted protein was collected. 所得的目的蛋白液中CRM197突变体的级分浓度可达到8mg/ml以上,纯度达到98%以上。 Level of the protein solution resulting mutant CRM197 content concentration can reach more than 8mg / ml, more than 98% purity. 实施例71、 CRM197突变体包涵体的溶解和变、复性,同实施例l。 Embodiment 71, CRM197 mutant variants and inclusion bodies were dissolved, renatured embodiment, with the embodiment of Example l. 2、 复性液的超滤分离和浓縮先用pH为10.0,浓度为100mmol/L的磷酸盐缓冲溶液对超滤器和大孔径(300kd)超滤膜进行平衡,保持进出口压力分别为1.3bar和0.3bar,开始进行大分子蛋白或杂质的截留浓縮。 2, separation and concentration by ultrafiltration refolding solution to a pH of 10.0, the concentration of 100mmol / L phosphate buffer solution to ultrafiltration and a large aperture (300 kD) ultrafiltration membrane balance, outlet pressure were held 1.3bar and 0.3bar, macromolecular protein concentrate interception started or impurities. 当复性缓冲液浓縮至1L左右时;用1一3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积:如此稀释2次,收集超滤透过液。 When the refolding buffer is concentrated to approximately 1L; refolding of the buffer solution was washed with 3 volumes of a 1, then the same volume of ultrafiltration: 2 dilution thus collected ultrafiltration permeate. 将大孔径(300kd)超滤膜堆换作小孔径(10kd) 超滤膜堆,按照上述方法将上述超滤透过液进行浓縮,浓縮至1L左右时,用2倍体积的上述磷酸盐缓冲液稀释后继续超滤至相同的体积,共稀释3次,收集浓縮液。 When the large pore size (300 kD) membrane stack change for small aperture (10 kD) ultrafiltration membrane reactor, according to the method described above was concentrated through ultrafiltration above was concentrated to approximately 1L, the phosphoric acid with 2 volumes of after dilution ultrafiltration continues salt buffer to the same volume, it was diluted 3 times, collecting the concentrate. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到80%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / ml, more than 80% purity. 3、 阴离子交换层析用300ml浓度为100mmol/L, pH为10. 0的磷酸盐平衡缓冲液平衡DEAE-S印harose FF (1.6X15cm)层析柱,将超滤后的浓縮液上柱。 3, anion-exchange chromatography with 300ml concentration of 100mmol / L, pH of phosphate buffer balanced equilibrium DEAE-S is printed harose FF 10. 0 (1.6X15cm) column, concentrating the solution after the ultrafiltration column . 4、 平衡、洗脱、过滤用浓度为100mmol/L, pH为10. 0的磷酸盐平衡缓冲液冲洗3个柱体积,然后将NaCl的浓度在20min内从0誦ol/L升至ljlOOmmol/L,继续在100min内将NaCl浓度从100誦ol/L升高到lmol/L进行梯度洗脱,收集洗脱蛋白液。 4, balance, elution with a concentration filtered 100mmol / L, pH balanced phosphate buffer 10.0 rinsed 3 column volumes, then the NaCl concentration from 0 recite in 20min ol / L was raised ljlOOmmol / L, will continue within 100min recite NaCl concentration was raised from 100 ol / L to lmol / L gradient elution, the protein was eluted. 所得的目的蛋白液中CRM197突变体的级分浓度可达到9mg/ml以上,纯度达到98%以上。 Level of the protein solution resulting mutant CRM197 content concentration can reach more than 9mg / ml, more than 98% purity. 实施例81、 CRM197突变体包涵体的溶解和变、复性,同实施例l。 Example 81, was dissolved and the CRM197 mutant variations, renaturation of inclusion bodies, with the embodiment of Example l. 2、 复性液的超滤分离和浓縮先用pH为ll.O,浓度为200咖ol/L的Tris-HCl缓冲溶液对超滤器和大孔径(500kd)超滤膜进行平衡,保持进出口压力分别为1.3bar和0.3bar,开始进行大分子蛋白或杂质的截留浓縮。 2, separation and concentration by ultrafiltration refolding solution to a pH of ll.O, coffee concentration of 200 ol / L of Tris-HCl buffer solution to ultrafiltration and a large aperture (500 kD) ultrafiltration membrane balance and maintain respectively, and outlet pressure 1.3bar 0.3bar, macromolecular protein concentrate interception started or impurities. 当复性缓冲液浓縮至1L左右时;用1一3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次,收集超滤透过液。 When the refolding buffer is concentrated to approximately 1L; refolding of the buffer solution was washed with 3 volumes of a 1, then the same volume of ultrafiltration; thus diluted 2 times, ultrafiltration permeate collected. 将大孔径(500kd)超滤膜堆换作小孔径(10kd)超滤膜堆,按照上述方法将上述超滤透过液进行浓縮,浓縮至1L左右时,用2倍体积的上述Tris-HCL缓冲液稀释后继续超滤至相同的体积,共稀释3次,收集浓縮液。 When the large pore size (500 kD) membrane stack change for small aperture (10 kD) ultrafiltration membrane reactor, according to the method described above was concentrated through ultrafiltration above was concentrated to approximately 1L, the above volumes of Tris 2 after dilution buffer -HCL ultrafiltration continued until the same volume, it was diluted 3 times, collecting the concentrate. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到80%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / ml, more than 80% purity. 3、 阴离子交换层析用300ml浓度为200mmol/L, pH为ll. O的Tris-HCL平衡缓冲液平衡QS印harose FF (1. 6 X15cm)层析柱,将超滤后的浓縮液上柱。 3, anion-exchange chromatography with 300ml concentration of 200mmol / L, pH to ll. O Tris-HCL is equilibrated with equilibration buffer QS printing harose FF (1. 6 X15cm) column, on the concentrate after ultrafiltration column. 4、 平衡、洗脱、过滤用浓度为200mmol/L, pH为11. 0的Tris-HCL平衡缓冲液冲洗3个柱体积,换用含有10mmol/L NaCl的pH为ll.O、浓度为200mmol/L Tris-HCL缓冲液作为洗脱液进行洗脱,收集洗脱蛋白液。 4, balance, elution with a concentration filtered 200mmol / L, pH to 11.0 balance of Tris-HCL buffer to wash with 3 column volumes, for a pH containing 10mmol / L NaCl for ll.O, 200mmol concentration / L Tris-HCL buffer eluted as eluant eluted protein was collected. 所得的目的蛋白液中CRM197突变体的级分浓度可达到10mg/ml以上,纯度达到98%以上。 Level of the protein solution resulting mutant CRM197 content concentration can reach more than 10mg / ml, more than 98% purity. 实施例91、 CRM197突变体包涵体的溶解和变、复性,同实施例l。 Example 91, CRM197 mutant variants dissolution and renaturation of inclusion bodies, with the embodiment of Example l. 2、 复性液的超滤分离和浓縮先用pH为8.0,浓度为500國ol/L的磷酸盐缓冲溶液对超滤器和大孔径(300kd)超滤膜进行平衡,保持进出口压力分别为1.3bar和0.3bar,开始进行大分子蛋白或杂质的截留浓縮。 2, separation and concentration by ultrafiltration refolding solution to a pH of 8.0, at a concentration of 500 States ol / L phosphate buffer solution to ultrafiltration and a large aperture (300 kD) ultrafiltration membrane balance and maintain outlet pressure respectively 1.3bar and 0.3bar, macromolecular protein concentrate interception started or impurities. 当复性缓冲液浓縮至1L左右时;用1一3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次,收集超滤透过液。 When the refolding buffer is concentrated to approximately 1L; refolding of the buffer solution was washed with 3 volumes of a 1, then the same volume of ultrafiltration; thus diluted 2 times, ultrafiltration permeate collected. 将大孔径(300kd)超滤膜堆换作小孔径(10kd) 超滤膜堆,按照上述方法将上述超滤透过液进行浓縮,浓縮至1L左右时,用2倍体积的上述磷酸盐缓冲液稀释后继续超滤至相同的体积,共稀释3次,收集浓縮液。 When the large pore size (300 kD) membrane stack change for small aperture (10 kD) ultrafiltration membrane reactor, according to the method described above was concentrated through ultrafiltration above was concentrated to approximately 1L, the phosphoric acid with 2 volumes of after dilution ultrafiltration continues salt buffer to the same volume, it was diluted 3 times, collecting the concentrate. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/ml,纯度达到80%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / ml, more than 80% purity. 3、 阴离子交换层析用300ml浓度为500mmol/L, pH为8. 0的磷酸盐平衡缓冲液平衡S0URCE30-Q( 1. 6X15cm) 层析柱,将超滤后的浓縮液上柱。 3, anion-exchange chromatography with 300ml concentration of 500mmol / L, pH balance (1. 6X15cm) S0URCE30-Q column balanced by phosphate buffer 8.0, the concentrated solution onto the column after the ultrafiltration. 4、 平衡、洗脱、过滤用浓度为500mmol/L, pH为8. 0的磷酸盐平衡缓冲液冲洗3个柱体积,换用含有O. 8mol/L NaCl的pH为8.0、浓度为500mmol/L磷酸盐缓冲液作为洗脱液进行洗脱,收集洗脱蛋白液。 4, balance, elution with a concentration filtered 500mmol / L, pH of the equilibration buffer 8.0 phosphate wash with 3 column volumes, containing a pH change O. 8mol / L NaCl 8.0, at a concentration of 500mmol / L phosphate buffer and eluted as eluant eluted protein was collected. 所得的目的蛋白液中CRM197突变体的级分浓度可达到8mg/ml以上,纯度达到98%以上。 Level of the protein solution resulting mutant CRM197 content concentration can reach more than 8mg / ml, more than 98% purity. 实施例IO1、 CRM197突变体包涵体的溶解和变、复性,同实施例l。 Example IO1 embodiment, the CRM197 mutant variants and dissolution and renaturation of inclusion bodies, with the embodiment of Example l. 2、 复性液的超滤分离和浓縮先用pH为8.0,浓度为1000mmol/L的Tris-HCL缓冲溶液对超滤器和小孔径(10kd)超滤膜进行平衡,保持进出口压力分别为L3bar和0.3bar,进行复性液的浓縮。 2, separation and concentration by ultrafiltration refolding solution to a pH of 8.0, a concentration of 1000mmol / L of Tris-HCL buffer solution to ultrafiltration and small aperture (10 kD) ultrafiltration membrane balance and maintain outlet pressure, respectively is L3bar and 0.3bar, concentrated renaturation solution. 当复性液浓縮至1L左右时;用1—3倍体积的上述缓冲液稀释复性液,再超滤到相同的体积;如此稀释2次,收集浓縮液。 When refolding solution was concentrated to about 1L; with the above buffer solution Refolding volumes of 1-3, and then to the same ultrafiltration volume; thus diluted twice concentrated solution was collected. 通过超滤浓縮收集的CRM197突变体的级分浓度可达到3mg/m1,纯度达到80%以上。 Collected and concentrated by ultrafiltration stage CRM197 mutant content concentration up to 3mg / m1, purity over 80%. 3、 阴离子交换层析用300ml浓度为1000mmol/L, pH为8. 0的Tris-HCL平衡缓冲液平衡DEAE-S印harose FF (1.6X15cm)层析柱,将超滤后的浓縮液上柱。 3, the concentration of anion-exchange chromatography with 300ml of 1000mmol / L, pH 8.0 as the buffer Tris-HCL balanced equilibrium DEAE-S plate harose FF (1.6X15cm) column, on the concentrate after ultrafiltration column. 4、 平衡、洗脱、过滤用浓度为1000mmol/L, pH为8. 0的Tris-HCL平衡缓冲液冲洗3个柱体积,换用含有lmol/LNaCl的pH为8. 0、浓度为1000mmol/L Tris-HCL缓冲液作为洗脱液进行洗脱,收集洗脱蛋白液。 4, balance, elution with a concentration filtered 1000mmol / L, pH balance of Tris-HCL buffer 8.0 wash 3 column volumes, containing a pH change lmol / LNaCl to 8.0, at a concentration of 1000mmol / L Tris-HCL buffer eluted as eluant eluted protein was collected. 所得的目的蛋白液中CRM197突变体的级分浓度可达到10mg/ml以上,纯度达到98%以上。 Level of the protein solution resulting mutant CRM197 content concentration can reach more than 10mg / ml, more than 98% purity.

Claims (9)

1. 一种CRM197突变体的纯化方法,包括如下步骤: (1)采用大肠杆菌以包涵体形式表达重组CRM197突变体,包涵体经过变、复性后得到含有目的蛋白组分的复性液; (2)复性液通过超滤进行分离和浓缩,并用Tris-HCl缓冲液进行换液,将复性液浓缩5-25倍,得超滤浓缩液; (3)上述超滤浓缩液通过阴离子交换层析进行纯化层析,截留核酸和大部分杂蛋白质,得到含有目的蛋白CRM197突变体的分级组分; (4)用平衡缓冲液对阴离子交换层析系统进行平衡,使目的蛋白吸附到阴离子层析柱上,大部分杂质穿出阴离子层析柱; (5)用添加了NaCl的平衡缓冲溶液对上述步骤(4)处理后的阴离子交换层析柱进行洗脱,使目的蛋白穿出阴离子交换层析柱,收集洗脱蛋白液,即得目的蛋白液。 A method of purifying CRM197 mutant thereof, comprising the steps of: (1) using E. coli expressing the recombinant CRM197 mutant, variant after inclusion, obtained after renaturation protein refolding solution containing a component in the form of inclusion bodies; (2) refolding solution and concentrated by ultrafiltration, and the medium was changed with Tris-HCl buffer, renaturation was concentrated 5-25 fold by ultrafiltration to obtain a concentrate; (3) the ultrafiltration concentrate was purified by anion exchange chromatography purified by chromatography, and most of the entrapped nucleic acid hybrid protein, to obtain a classified component comprising CRM197 mutant protein; and (4) with the equilibration buffer for the anion exchange chromatography systems to balance the protein adsorbed to anionic chromatographic column, most of the impurities piercing anion chromatography column; (5) supplemented with NaCl in the equilibration buffer solution of the above step (4) the anion exchange chromatography column eluted process, piercing anionic target protein exchange chromatography column, eluted protein was collected, to obtain the protein solution.
2、 如权利要求1所述的CRM197突变体的纯化方法,其特征在于步骤(2)中所述的复性液通过超滤进行分离和浓縮,是一次性进行超滤浓縮。 2. The purification process according to claim 1 CRM197 mutant claim, wherein the step (2) compound was isolated and the concentration by ultrafiltration, is disposable and concentrated by ultrafiltration.
3、 如权利要求1所述的CRM197突变体的纯化方法,其特征在于步骤(2)中所述的复性液通过超滤进行分离和浓縮,是先超滤去除大分子蛋白或杂质,再进行超滤浓縮。 3, the purification process as claimed in claim 1 CRM197 mutant claim, wherein the step (2) compound was isolated and the concentration by ultrafiltration, ultrafiltration is to remove protein molecules or impurities, then concentrated by ultrafiltration.
4、 如权利要求1所述的CRM197突变体的纯化方法,其特征在于所述步骤(3)中所述的阴离子交换层析,所使用的阴离子交换层析柱的层析介质选自琼脂糖凝胶类或聚苯乙烯类,配基选自季铵基或二乙基氨乙基。 4. The purification process according to claim 1 CRM197 mutant claims, wherein said step (3) in the anion exchange chromatography, anion exchange chromatography using a column of Sepharose medium is selected from gels or polystyrene with quaternary ammonium group or a group selected from diethylaminoethyl.
5、 如权利要求1或4所述的CRM197突变体的纯化方法,其特征在于所述步骤(3)中所述的阴离子交换层析,所使用的阴离子交换层析介质选自下列之一:DEAE-S印harose FF, QS印harose FF, S0URCE30-Q或Q-Big-Beds。 5, CRM197 as claimed in claim 1 or 4, wherein the purification process mutant, wherein said step (3) in the anion exchange chromatography, the anion exchanger used chromatography media selected from one of the following: DEAE-S plate harose FF, QS printing harose FF, S0URCE30-Q or Q-Big-Beds.
6、 如权利要求1或4所述的CRM197突变体的纯化方法,其特征在于所述步骤(3)中所述的阴离子交换层析,所使用的阴离子交换层析介质DEAE-S印harose FF层析介质。 6. The purification method as claimed in claim 4 or 1 CRM197 mutant claims, wherein said step (3) in the anion exchange chromatography, anion exchange chromatography using DEAE-S printing medium harose FF chromatography media.
7、 如权利要求1所述的CRM197突变体的纯化方法,其特征在于所述步骤(4)和(5) 中的平衡缓冲液选用Tris-HCl缓冲液或磷酸盐缓冲液,缓冲液的浓度为10國ol/L-lmol/L; 缓冲液的pH值为6. 0-11.0。 7. The purification process according to claim 1 CRM197 mutant buffer concentration claims, characterized in that said step (4) and (5) the selection of the equilibration buffer Tris-HCl buffer or phosphate buffer, State 10 ol / L-lmol / L; pH value of the buffer 6. 0-11.0.
8、 如权利要求1所述的CRM197突变体的纯化方法,其特征在于所述步骤(5)中所述的添加了NaCl的平衡缓冲液,其中NaCl浓度为10mmol/L—lmol/L。 8. The purification process according to claim 1 CRM197 mutant claims, wherein said step (5) in the equilibration buffer was added the NaCl, wherein the NaCl concentration of 10mmol / L-lmol / L.
9、 如权利要求1所述的CRM197突变体的纯化方法,其特征在于所述步骤(5)中的洗脱方式采用非梯度洗脱或连续梯度洗脱。 9. The purification process according to claim 1 CRM197 mutant claim, wherein the step elution embodiment (5) of the non-continuous gradient elution or gradient elution.
CN 200710013378 2007-03-13 2007-03-13 Method for purifying CRM197 mutant CN101265288B (en)

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